A cell culture plate for organoid culture and a method of manufacturing the same

Abstract

The application belongs to the technical field of cell culture and specifically relates to a cell culture plate for organoid culture and a preparation method thereof, which is formed by physical adhesion of a coating solution to the surface of the cell culture plate and solidification. The coating solution comprises: polymer A, which is a hydrophilic material with a hydrophilic-lipophilic balance value greater than 16; polymer B, which is a polyether substance; and polymer C, which is a non-ionic surfactant; wherein the weight ratio of the three is 1-10:0.1-0.2:0.1-0.5; the cell culture plate is a polystyrene culture plate; and the water contact angle of the coating is 10-60°. Compared with the prior art, the application solves the problem that the coating can be attached only by chemical cross-linking, surface activation or surface grafting in the prior art. The ultra-low adsorption cell culture plate has the following advantages: ultra-low cell adhesion; simple preparation process; no chemical cross-linking agent residue; stable physical and chemical properties; and compatibility with the existing standard production process of the culture plate material.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a cell culture plate for organoid culture and its preparation method. Background Technology

[0002] Organoids are three-dimensional self-organizing structures derived from stem cells or primary tissue cells, and are widely used in tumor research, drug screening, toxicology evaluation, disease model construction, and regenerative medicine research. During the culture process, organoids rely on a culture microenvironment with good suspension and extremely low adhesion, especially the surface characteristics of the culture plate, which have a decisive influence on the formation efficiency, size uniformity, and structural integrity of organoids.

[0003] Traditional methods for preparing ultra-low adsorption culture plates mainly include: (1) A hydrogel coating is formed by chemical crosslinking of polymer materials such as acrylamide and polyethylene glycol (PEG). For example, a superhydrophilic coating for cell culture and its preparation method disclosed in CN112480750A uses chitosan and polyvinyl alcohol to crosslink and construct an interpenetrating network. For example, the cell culture system, its method and its use disclosed in CN117098819A form a matrix suitable for cell growth through an alternating layer structure of hydrophilic polymers and polyelectrolytes. (2) The surface energy of polystyrene (PS) is changed by plasma treatment or ultraviolet crosslinking. For example, CN119193490A discloses a coating liquid and treatment method and culture material for ultra-low adsorption cell culture surface. First, the surface of the material to be treated is subjected to low temperature plasma treatment and then coated with coating liquid. For example, CN103087916A discloses a cell culture plate coated with Matrigel and its preparation method and application. After plasma treatment, Matrigel is connected to the surface of polystyrene cell culture plate or culture dish by chemical riveting technology in the form of covalent bonds. (3) Increase the surface hydrophilic layer by chemical methods such as surface grafting, such as the preparation method and application of a cell culture plate coated with carboxymethyl cellulose coupled with RGD short peptide disclosed in CN103966097A, and introduce polypeptides by grafting on the surface of the culture plate, such as the cell culture material modified by blending and its preparation method disclosed in CN104151591A, and modify the cell culture material by carboxylated styrene-butadiene rubber doping by blending, etc.

[0004] However, these methods suffer from problems such as complex processes, high costs, residual chemical cross-linking agents, insufficient coating stability, or interference with cell growth. Furthermore, while some commercial organoid culture plates provide a good suspension environment, their coating materials are not suitable for all cell types, and some products are expensive, hindering widespread use.

[0005] Therefore, there is an urgent need for an ultra-low adsorption culture plate technology that is simple to prepare, low in cost, highly stable, non-chemically cross-linked, does not interfere with organoid growth, and can be implemented in batches on standard PS production lines. Summary of the Invention

[0006] The purpose of this invention is to provide a cell culture plate for organoid culture and its preparation method to solve at least one of the aforementioned problems, thereby addressing the issue in existing technologies where coatings require chemical cross-linking, surface activation, or surface grafting. This solution utilizes the physical adsorption of hydrophilic polymers rather than chemical reactions to form an ultra-low adsorption cell culture plate, which features: ultra-low cell adhesion; simple preparation process; no chemical cross-linking agent residue; stable physicochemical properties; and compatibility with existing standard production processes for culture plates.

[0007] The objective of this invention is achieved through the following technical solution: The first aspect of this invention discloses a cell culture plate for organoid culture, wherein a coating liquid is physically attached to the surface of the cell culture plate and then cured to form a coating. The coating fluid includes: Polymer A is a hydrophilic material with a hydrophilic-lipophilic balance value greater than 16; Polymer B is a polyether-based substance; Polymer C is a nonionic surfactant; The weight ratio of polymer A, polymer B and polymer C is 1-10:0.1-0.2:0.1-0.5. The cell culture plate mentioned is a polystyrene culture plate; The water contact angle of the coating is 10-60°.

[0008] Preferably, polymer A is polyvinyl alcohol with a hydrophilic-lipophilic balance value greater than 16.

[0009] Preferably, polymer B is polyethylene glycol.

[0010] Preferably, the polymer C is polysorbate.

[0011] More preferably, the coating liquid comprises: polyvinyl alcohol, polyethylene glycol and polysorbate with a hydrophilic-lipophilic balance value greater than 16, in a weight ratio of 1:0.1:0.1.

[0012] Preferably, the coating solution further includes 0.1-0.5 wt% of a first auxiliary agent of the total polymer amount, wherein the first auxiliary agent is a polyol and / or glycerol.

[0013] Preferably, in the coating solution, the weight ratio of polymer A, polymer B and polymer C is 1:0.1:0.1, polymer A is polyvinyl alcohol with a hydrophilic-lipophilic balance value greater than 16, polymer B is polyethylene glycol, and polymer C is polysorbate.

[0014] Preferably, the thickness of the coating is 0.1-100 μm.

[0015] A second aspect of this invention discloses a method for preparing a cell culture plate for organoid culture as described in any of the above descriptions, comprising the following steps: S1: Prepare the coating solution; S2: Add the coating solution prepared in step S1 into the wells of the cell culture plate and spread the coating solution evenly on the inner wall of the well, and then remove the excess coating solution. S3: Heat and dry the cell culture plate obtained in step S2 to solidify the coating solution and physically adhere it to the surface of the cell culture plate. S4: Sterilize the cell culture plate obtained by solidification in step S3; In step S3: the coating solution is solidified and physically adhered to the surface of the cell culture plate by heating and drying at a temperature of 25-80℃ for 0.5-4 hours.

[0016] Preferably, it includes one or more of the following: i) In step S2: the volume of coating solution added to each well of the cell culture plate is 200-300 μL / well; ii) In step S2: the coating liquid is evenly spread on the inner wall of the hole by spin coating at 500-3000 rpm; iii) In step S3: the coating solution is solidified on the surface of the cell culture plate by means of forced air drying or vacuum drying.

[0017] A third aspect of the present invention discloses the use of a cell culture plate for organoid culture as described above in the production of cell culture plates for culturing organoids, stem cell spheres, spheroids derived from induced pluripotent stem cells, or three-dimensional cell clusters.

[0018] The working principle of this invention is as follows: This cell culture plate forms a hydrophilic gel-like coating with interface regulation function on the substrate surface. During in vitro culture, cells typically rely on exocrine matrix proteins or adhesion proteins in the culture medium to adsorb onto the substrate surface and form anchorage points, thus achieving adherent growth. The hydrophilic coating constructed in this method weakens the protein-mediated adhesion interface between cells and the substrate. Due to the lack of stable adhesion anchoring structures, cells struggle to spread and adhere to the substrate surface, thus spontaneously aggregating to form three-dimensional cell spheres or organoid structures driven by gravity and intercellular interactions.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This solution utilizes a non-crosslinked hydrophilic polymeric physical adsorption coating. The coating is composed of hydrophilic, water-soluble polymers A, B, and C, forming an interface-controlled coating. The preparation of this coating does not involve crosslinking reactions, produces no chemical residues, and offers higher safety for cell culture. This coating is soluble in boiling water; possesses good hydrophilicity; can form a uniform, transparent film; has a certain adhesion ability, forming a stable physical adhesion with PS (cell culture plates); has high biocompatibility; contains repeating structural units that can form a stable hydrogen bond network with water molecules; and the solution system can be cured into a film on the substrate surface through physical drying methods such as evaporation or water bath.

[0020] 2. This method utilizes a stable hydrophilic layer formation mechanism to form a continuous and stable adsorption layer on the surface of the cell culture plate by controlling the drying temperature, time, and concentration of the coating solution. Based on weak physical adsorption and film solidification for stable adhesion, it eliminates the need for additional crosslinking agents or photoinitiators, which could significantly reduce the probability of cell adhesion and introduce no chemical residues.

[0021] 3. The coating proposed in this solution has high compatibility and low cost, and the process can be fully adapted to existing injection molding and board making production lines without the need for additional equipment. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the preparation process of ultra-low adsorption cell culture plates.

[0023] Figure 2 The results show the observation of cell microspheres in cell culture plates prepared with different formulations in the specific implementation method.

[0024] Figure 3 This is a side cross-sectional view of several typical cell culture plates after coating with the coating prepared in Example 1.

[0025] Figure 4 Cross-sectional scanning electron microscope (SEM) images of cell culture plates before and after coating (using the coating prepared in Example 1).

[0026] Figure 5 The water contact angles of the cell culture plate before and after coating (using the coating prepared in Example 1).

[0027] Figure 6 This is a bright-field 10X image of cell microspheres cultured in a cell culture plate in Example 5.

[0028] Figure 7 This is a comparison diagram showing the differences between cells cultured in cell culture plates of Example 1, Control Example, and Comparative Example 1. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments or drawings of the present invention are merely for illustrating the present invention and are not intended to limit the present invention.

[0030] Unless otherwise specified, the reagents used in the following description are conventional commercial products, the methods used are common knowledge in the field, and any other matters not covered herein can be handled using existing technology.

[0031] This invention discloses an ultra-low adsorption cell culture plate applicable to the culture of three-dimensional spheres such as organoids and stem cells, and its preparation method.

[0032] This cell culture plate uses polystyrene (PS) as the substrate. By coating the inner surface of the wells with the coating solution described in this invention and then physically drying it, hydrophilic polymers adhere to the PS surface through physical adsorption, forming a stable hydrophilic non-adhesive coating. The preparation process does not involve chemical crosslinking, photocrosslinking, or surface grafting, avoiding the problem of residual chemical groups that may adversely affect cells in traditional surface treatment methods. The prepared ultra-low adsorption cell culture plate can effectively inhibit cell adhesion behavior and improve the formation efficiency of three-dimensional cell clusters such as spheroids and organoids, thereby ensuring its stability and reproducibility in applications such as drug screening, disease model construction, and stem cell research. This invention offers advantages such as simple preparation process, low cost, and strong compatibility, and can be adapted to various cell culture plate types, including U-shaped, V-shaped, flat-bottomed, and other types, making it suitable for large-scale production.

[0033] More specifically, the coating solution of the present invention comprises: coating polymer A is a hydrophilic material with a hydrophilic-lipophilic balance (HLB) greater than 16, such as polyvinyl alcohol (PVA); coating polymer B is a polyether substance, such as polyethylene glycol (PEG); and coating polymer C is a nonionic surfactant, such as polysorbate. It is obtained by forming a film from an aqueous solution followed by physical drying (such as natural evaporation or low-temperature drying), forming a continuous thin film coating of hydrophilic water-soluble coating material on the surface of a polystyrene-based cell culture plate. This coating does not contain crosslinking agents, photoinitiators, or other reactive components. After drying and curing, the formed coating exhibits ultra-low cell adhesion under culture conditions, promoting cell suspension and aggregation in the well bottom region; the thickness of the coating is 0.1-100 μm.

[0034] If necessary, 0.1-0.5 wt% of the total polymer (total of polymer A + polymer B + polymer C) of polyol, glycerol, or a combination thereof may be added to the coating solution to improve the flexibility and durability of the coating, reduce film-forming stress, and enhance crosslinking stability.

[0035] The coating solution is applicable to cell culture plates with U-shaped, V-shaped, round, or flat bottom pores.

[0036] The water contact angle of the coating in this design is 10-60°.

[0037] More specifically, the method for attaching the coating solution of the present invention to the surface of a cell culture plate and constructing a coating includes the following steps, such as... Figure 1 As shown: (1) Prepare the coating solution (coating solution) according to the formula; (2) Add the coating solution to the wells of the PS cell culture plate; (3) Spread the coating liquid evenly on the bottom and sidewalls of the plate holes by shaking, spinning or dipping. (4) Discard the excess coating solution; (5) Heat and dry the cell culture plate at 25-80℃ for 0.5-4 h to form a continuous solidified film on the polystyrene surface; the solidified film is stably attached to the PS surface by physical adsorption, without involving chemical grafting or cross-linking reaction. (6) Sterilize the cell culture plates that have formed the coating.

[0038] In step (2), the volume of coating solution added to each well of the cell culture plate is 200-300 μL / well (96-well plate).

[0039] In step (3), a spin coating operation of 500-3000 rpm is used to obtain a uniform coating with a coating thickness between 0.1-100 μm.

[0040] In step (5), the drying method is hot air oven, forced air drying, vacuum drying or a combination thereof.

[0041] In step (6), after the coating is formed, the uncoated material is removed by ultrasonic cleaning or multiple sterile water rinsing.

[0042] Cell culture plates coated with this material can improve the formation efficiency and size uniformity of tumor organoids, stem cell spheres, induced pluripotent stem cell-derived spheroids, or other three-dimensional cell clusters. They are particularly suitable for the construction of scaffold-free organoids, stem cell spheres, 3D multicellular spheroids, and related three-dimensional cell models, and are an important technological platform in the field of novel three-dimensional biological models.

[0043] The cell culture plates coated with this material are formed through physical adsorption of hydrophilic polymers rather than chemical reactions. They have the following characteristics: ultra-low cell adhesion; simple preparation process; no chemical cross-linking agent residue; applicable to various three-dimensional culture models such as organoids and stem cell spheres; and compatible with existing standard production processes for culture plates.

[0044] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention fall within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0045] Examples 1-4 Examples 1-4 provide several representative coating solutions for ultra-low adsorption cell culture plates (Formulations 1-4 correspond to Examples 1-4 respectively), and the formulations are shown in Table 1 below.

[0046] Table 1. Coating solution formulations for Examples 1-4 The formulations in Examples 1-4 were prepared by the following steps to form a coating and physically adhere it to the surface of the cell culture plate: (1) Prepare several representative coating solutions according to formulas 1-4 respectively; (2) Add the coating solution into the culture container (the well of the cell culture plate, specifically a standard polystyrene 96-well U-shaped bottom plate cell culture plate). The surface of the cell culture plate does not need to be pretreated. (3) Add 200-300 μL of coating liquid to each well and spread the coating material evenly on the bottom and sidewalls by shaking, spin coating or dip coating. In this embodiment, it is preferred to spread the coating material evenly by spin coating at 1000 rpm or by letting it stand. In other embodiments, the spin coating speed can be selected in the range of 500-3000 rpm, which can meet the requirements of even spreading. (4) Discard the excess coating solution; (5) Place the culture container in a (vacuum) oven or forced-air drying oven and heat the culture container at 60°C for 2 hours to cure the coating; in other embodiments, it is also optional to heat and dry at 25-80°C for 0.5-4 hours to ensure that the coating can be dried evenly. (6) Rinse with sterile water 1-3 times to remove the coating solution that has not formed a film, and then sterilize using gamma rays, ultraviolet light or electron beam irradiation according to the standard procedure.

[0047] like Figure 2 The image shows photographs of MCF-7 cell microspheres cultured in cell culture plates after coating with coating solution for several formulations in Examples 1-4. As shown in the figure, it can be seen that the cell microspheres of formulations 2, 3, and 4 did not completely aggregate. Therefore, formulation 1 (corresponding to Example 1) has the best effect.

[0048] Figure 3 This is a side cross-sectional view of several typical cell culture plates coated with the formulation of Example 1, further combined with... Figure 4 The SEM image of the cross-section of the culture container (with coating) prepared in Example 1 shown shows that the coating thickness is approximately 20 μm.

[0049] Figure 5 The figure shows a comparison of the water contact angles of the culture container (including coating) prepared in Example 1 before and after coating. It can be seen from the figure that the water contact angle before coating is 68° and the water contact angle after coating is 34°, indicating that the hydrophilicity of the container is greatly increased after coating.

[0050] Example 5 Organoid culture was performed using cell culture plates. First, standard 96-well U-bottom plates without any treatment were used to prepare the culture plates. Functionalized coated plates were then prepared according to the formulation and method in Example 1, and subsequently incubated at 37°C with 5% CO2 for 10-30 min to equilibrate. Cells were digested, and a cell suspension was prepared at 5000 cells / 100 μL. The cell suspension was seeded onto the coated plate surface at 100 μL / well, allowing the cells to spontaneously aggregate at the bottom of the wells under gravity. On day 2, the cells began to self-assemble into cell microspheres (MCF-7), at which point 100 μL of the corresponding cell culture medium was added. Culture was continued for 3-14 days, with the culture medium changed every 1-3 days.

[0051] Figure 6 The image shows cell microspheres (MCF-7) cultured using a standard layout ultra-low adsorption cell culture plate. As shown, the cell culture plate is numbered by row (AH) and column (1-12), with rows A and H, and columns 1 and 12 filled with phosphate-buffered saline (PBS) to reduce culture medium evaporation. Wells B2-G11 are for cell microsphere culture. The image demonstrates high success rate and good uniformity of the cell microspheres.

[0052] Example 6 The cell culture plates from Examples 1-4 were cultured with MCF-7 cell microspheres according to the method in Example 5. After 2 days of culture, the average particle size of each cell sphere (the pseudo-spheroid of the cell) in the 96-well plate was measured, calculated as (diameter at longest point + diameter at shortest point) / 2. The average particle size of the total number of cell spheres in each group was determined, along with the coefficient of variation (CV) = σ / μ × 100%, where σ is the standard deviation and μ is the average. This indicates that the CV value of the cell sphere particle size in Examples 1-4 was <10%, demonstrating that each group of cells stably formed uniformly sized pseudo-spheroids after culture. The results are shown in Table 2 below.

[0053] Table 2 Statistical results of Example 6 Comparative Example 1 This comparative example provides a coating solution with the following formulation: 3 g PEG and 97 g deionized water. The raw materials were mixed according to the formulation and filtered to obtain the coating solution; cell culture plates were prepared according to the method in Example 1.

[0054] Comparison Example This comparative example provides a blank control with the following formulation: 100 g of deionized water. Cell culture plates were prepared according to the method in Example 1.

[0055] Subsequently, the cell culture plates were placed in a 37°C, 5% CO2 incubator for equilibration for 10-30 min; the cells were digested and cell suspensions were prepared; the cell suspensions were then seeded onto cell culture plates of Formula 1, Control Example, and Comparative Example 1, respectively. Figure 7 Images of cells cultured on the three cell culture plates are shown in the figure. Cells adhered to the cell culture plates of Comparative Example 1 and Control Example, but did not adhere to the cell culture plate of Formula 1.

[0056] Example 7 This embodiment further adds 0.1 wt% of the total polymer amount of glycerol to the formula of Example 1 (Formula 1).

[0057] Tests have shown that the cell culture plates prepared according to the formulation of this embodiment have similar performance to those prepared in Example 1, and will not be described in detail here.

[0058] Example 8 This embodiment adds 0.5 wt% of the total polymer amount of glycerol to the formula of Example 1 (Formula 1).

[0059] Tests have shown that the cell culture plates prepared according to the formulation of this embodiment have similar performance to those prepared in Example 1, and will not be described in detail here.

[0060] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A cell culture plate for organoid culture, characterized in that, The coating solution physically adheres to the surface of the cell culture plate and solidifies to form a coating. The coating fluid includes: Polymer A is a hydrophilic material with a hydrophilic-lipophilic balance value greater than 16; Polymer B is a polyether-based substance; Polymer C is a nonionic surfactant; The weight ratio of polymer A, polymer B and polymer C is 1-10:0.1-0.2:0.1-0.

5. The cell culture plate mentioned is a polystyrene culture plate; The water contact angle of the coating is 10-60°.

2. The cell culture plate for organoid culture according to claim 1, characterized in that, The polymer A is polyvinyl alcohol with a hydrophilic-lipophilic balance value greater than 16.

3. A cell culture plate for organoid culture according to claim 1, characterized in that, The polymer B is polyethylene glycol.

4. A cell culture plate for organoid culture according to claim 1, characterized in that, The polymer C is polysorbate.

5. A cell culture plate for organoid culture according to claim 1, characterized in that, The coating solution further includes 0.1-0.5 wt% of a first auxiliary agent of the total polymer amount, wherein the first auxiliary agent is a polyol and / or glycerol.

6. A cell culture plate for organoid culture according to claim 1, characterized in that, In the coating solution, the weight ratio of polymer A, polymer B, and polymer C is 1:

1. 0.1:0.1, Polymer A is polyvinyl alcohol with a hydrophilic-lipophilic balance value greater than 16, Polymer B is polyethylene glycol, and Polymer C is polysorbate.

7. A cell culture plate for organoid culture according to claim 1, characterized in that, The thickness of the coating is 0.1-100 μm.

8. A method for preparing a cell culture plate for organoid culture as described in any one of claims 1-7, characterized in that, Includes the following steps: S1: Prepare the coating solution; S2: Add the coating solution prepared in step S1 into the wells of the cell culture plate and spread the coating solution evenly on the inner wall of the well, and then remove the excess coating solution. S3: Heat and dry the cell culture plate obtained in step S2 to solidify the coating solution and physically adhere it to the surface of the cell culture plate. S4: Sterilize the cell culture plate obtained by solidification in step S3; In step S3: the coating solution is solidified and physically adhered to the surface of the cell culture plate by heating and drying at a temperature of 25-80℃ for 0.5-4 hours.

9. A method for preparing a cell culture plate for organoid culture according to claim 8, characterized in that, Includes one or both of the following: i) In step S2: the volume of coating solution added to each well of the cell culture plate is 200-300 μL / well; ii) In step S2: the coating liquid is evenly spread on the inner wall of the hole by spin coating at 500-3000 rpm.

10. A method for preparing a cell culture plate for organoid culture according to claim 8, characterized in that, In step S3: the coating solution is solidified on the surface of the cell culture plate to form a coating by means of forced air drying or vacuum drying.

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